The Structure of Proteins
Enzymes are globular proteins and quiet compact due to the considerable amount of folding of the long polypeptide chain. The polypeptide chain essentially consists of the "backbone" and its "side-groups". As the peptide bond is planar, only rotations around the C.sub..alpha. --N axis and the C.sub..alpha. --C.sup.' axis are permitted. Rotation around the C.sub..alpha. --N bond of the peptide backbone is denoted by the torsion angle .phi. (phi), rotation around the C.sub..alpha. --C' bond by .psi. (psi) vide, e.g. Creighton, T. E. (1984), Proteins; W. H. Freeman and Company, New York!. The choice of the values of these angles of rotation is made by assigning the maximum value of +180.degree. (which is identical to +180.degree.) to the maximally extended chain. In the fully extended polypeptide chain, the N.sub.1 C.sub..alpha. and C' atoms are all "trans" to each other. In the "cis" configuration, the angles .phi. and .psi. are assigned the value of 0.degree.. Rotation from this position around the bonds, so that the atoms viewed behind the rotated bond move "counterclockwise", are assigned negative values by definition, those "clockwise" are assigned positive values. Thus, the values of the torsion angles lie within the range -180.degree. to +180.degree..
Since the C.sub.60 -atoms are the swivel point for the chain, the side-groups (R-groups) associated with the C.sub.60 -atoms become extremely important with respect to the conformation of the molecule.
The term "conformation" defines the participation of the secondary and tertiary structures of the polypeptide chains in moulding the overall structure of a protein. The correct conformation of a protein is of prime importance to the specific structure of a protein and contributes greatly to the unique catalytic properties (i.e. activity and specificity) of enzymes and their stability.
The amino acids of polypeptides can be divided into four general groups: nonpolar, uncharged polar, and negatively or positively charged polar amino acids. A protein molecule, when submerged in its aqueous environment in which it normally occurs, tends to expose a maximum number of its polar side-groups to the surrounding environment, while a majority of its nonpolar sidegroups are oriented internally. Orientation of the side-groups in this manner leads to a stabilization of protein conformation.
Proteins, thus, exist in a dynamic equilibrium between a folded and ordered state, and an unfolded and disordered state. This equilibrium in part reflects the short range interactions among the different segments of the polypeptide chain, which tends to stabilize the overall structure of proteins. Thermodynamic forces simultaneously tend to promote randomization of the unfolding molecule.
A way to engineer stabilized proteins is to reduce the extend of unfolding by decreasing the flexibility of the polypeptide backbone, and simultaneously decreasing the entropy of the unfolded chain. So far only few attempts have been made to implement this rationale in the development of novel stabilized enzymes.
A genera principle of increasing protein thermostability has been provided Suzuki, Y. (1989); Proc. Japan Acad.; 65 Ser. B!. In this article Suzuki states that the thermostability of a globular protein can be enhanced cumulatively to a great extent by increasing the frequency of proline occurrence at the second site of .beta.-turns without significant alterations in the secondary and tertiary structures as well as in the catalytic function of enzymes. The principle is based on various facts and findings, among these the fact the proline residues show a strong tendency to occur preferentially at the second site of .beta.-turns Levitt, M (1978); Biochemistry; 17 4277-4285; and Chou, P. Y. & Fasman, G. D. (1977); J. Mol. Biol.; 115 135-175!. The principle is restricted to insertion of proline into the second site of 62-turns in proteins, no other sites are mentioned.
International Patent Publication WO 08/01520 (Cetus Corporation, USA) provides a method for increasing the stability of a protein by decreasing the configurational entropy of unfolding the protein. The method is applied on a Streptomyces rubiqinosus xylose isomerase, and it involves substitution of an amino acid with proline, or replacement of glycine with alanine, at predicted substitution sites.
It is an object of this invention to provide novel enzymes having improved stability.